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Host-Feeding Patterns of Culex pipiens and Other PotentialMosquito Vectors (Diptera: Culicidae) of West Nile Virus(Flaviviridae) Collected in PortugalAuthor(s): Hugo Costa Osório, Líbia Zé-Zé and Maria João AlvesSource: Journal of Medical Entomology, 49(3):717-721. 2012.Published By: Entomological Society of AmericaDOI: http://dx.doi.org/10.1603/ME11184URL: http://www.bioone.org/doi/full/10.1603/ME11184

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VECTOR/PATHOGEN/HOST INTERACTION, TRANSMISSION

Host-Feeding Patterns of Culex pipiens and Other Potential MosquitoVectors (Diptera: Culicidae) of West Nile Virus (Flaviviridae)

Collected in Portugal

HUGO COSTA OSORIO,1 LIBIA ZE-ZE, AND MARIA JOAO ALVES

Centre for Vectors and Infectious Diseases Research, National Institute of Health, Av. da Liberdade n� 5,2965-575 Aguas de Moura, Portugal

J. Med. Entomol. 49(3): 717Ð721 (2012); DOI: http://dx.doi.org/10.1603/ME11184

ABSTRACT The host blood-feeding patterns of mosquito vectors affects the likelihood of humanexposure to zoonotic pathogens, including West Nile Virus (family Flaviviridae, genus Flavivirus,WNV). In Portugal, data are unavailable regarding the blood-feeding habits of common mosquitospecies, including Culex pipiens L., considered the primary vector of WNV to humans. The sourcesof bloodmeals in 203 blood-fed mosquitoes of nine species collected from June 2007 to November 2010in 34 Portuguese counties were analyzed by sequencing cytochrome-b partial fragments. Cx. pipienswas themostcommonspeciescollectedandsuccessfully analyzed(n�135/78). Inaddition,blood-fedfemales of the following species were analyzed: Ochlerotatus caspius Pallas (n � 20), Culex theileriTheobald (n� 16),Anopheles maculipennis s.l. Meigen (n� 10),Culiseta longiareolataMacquart (n�7), Aedes aegypti L. (n � 6), Culex perexiguus Theobald (n � 3), Culiseta annulata Schrank (n � 3),and Ochlerotatus detritus Haliday (n � 3). The Cx. pipiens mosquitoes fed predominantly on birds(n � 55/78, 70.5%), with a high diversity of avian species used as hosts, although human blood wasidentiÞed in 18 specimens (18/78, 23.1%). No signiÞcant differences were found between thehost-feeding patterns of blood-fedCx. pipiens collected in residential and nonresidential habitats. Theoccurrence of human derived blood meals and the presence of a mix avianÐhuman bloodmealaccordingly suggest this species as a potential vector of WNV. Therefore, in Portugal, Cx. pipiensmayplay a role both in the avian-to-avian enzootic WNV cycle and in the avian-to-mammal transmission.In this context, the identity of Cx. pipiens (considering the forms molestus and pipiens) and thepotential consequence on feeding behavior and WNV transmission are discussed.

KEY WORDS mosquito bloodmeal identiÞcation, Culex pipiens, West Nile virus

The host-feeding patterns of mosquitoes are driven byseveral factors, including innate tendencies, host avail-ability and abundance, host defensive behaviors, andßight behavior and feeding periodicity of mosquitoes(Clements 1999). Different vector species have dif-ferent host-feeding patterns that may expose humansto multihost zoonotic vector-borne pathogens (Kil-patrick et al. 2007, Molaei et al. 2008). The emergence,dispersion, and maintenance of a vector-borne patho-gen are affected by the efÞciency of transmission,which depends on the convergence in time and spacebetween competent vectors, competent vertebratehosts, and the pathogen. Therefore, the blood-feedingbehaviors of mosquito vectors play a critical role in thetransmission and maintenance of vector-borne patho-gens in natural systems (Kent 2009).Culex pipiens L. is one of the most ubiquitous mos-

quito species in the world (Farajollahi et al. 2011), andbased on our surveillance results obtained from 2005throughout 2010, it is one of the most frequent and

widespread mosquito collected in residential and non-residential areas in Portugal (Osorio et al. 2010). Itconsists of two forms, molestus and pipiens, that aremorphologically indistinguishable but exhibit impor-tant behavioral and physiological differences and oc-cupy different habitats in the northern regions of Eu-rope, underground and aboveground, respectively(Vinagradova 2000). In some regions of Portugal,physiological, behavioral, and genetic data provideevidence for the sympatric occurrence of both forms(Gomes et al. 2009).

The Cx. pipiens complex represents the primaryenzootic vector of West Nile virus (family Flaviviri-dae, genus Flavivirus,WNV) in Europe (Hubalek andHalouzka 1999) and northeastern and north centralUnited States (Apperson et al. 2004, Molaei et al. 2006)and also is considered a potential epidemic vector(Hamer et al. 2008). However, since the detection ofWNV in North America in 1999 (Anderson et al. 1999),�60 mosquito species have tested positive for WNV(see Centers for Disease Control [CDC], West Nilevirus homepage at http://www.cdc.gov/ncidod/dvbid/1 Corresponding author, e-mail: hugo.osorio@insa.min-saude.pt.

0022-2585/12/0717Ð0721$04.00/0 � 2012 Entomological Society of America

westnile/), and several species also have been impli-cated as potential secondary vectors because of theirlocal abundance, vector competence in the laboratory(Turell et al. 2005), and frequent reports of infectionwith WNV in nature (Andreadis et al. 2004, Appersonet al. 2004).

In Portugal, WNV was isolated from three mosquitospeciesÑAnopheles maculipennis s.l. Meigen, CulexperexiguusTheobald, andCx. pipiens (Filipe and Pinto1972, Parreira et al. 2007)Ñalthough the precise rolethat each species plays in the enzootic transmissionamong birds or epidemic transmission to humans is notpresently clear. To our knowledge, this is the Þrststudy on feeding behavior of wild mosquitoes col-lected in Portugal. Regarding transmission of WNV,we assessed the blood-feeding patterns of some rep-resentative mosquito species in Portugal, namely,Aedes aegypti L. (Madeira Island), An. maculipennis,Cx. perexiguus, Culex theileri Theobald, Culiseta annu-lata Schrank, Culiseta longiareolata Macquart, Ochle-rotatus caspius Pallas, Ochlerotatus detritus Haliday,and particularlyCx. pipiens.The role of this species onWNV transmission is questioned considering the pres-ence of the forms pipiens and molestus and their hostpreference.

Materials and Methods

Mosquito Collection and Species Identification.Mosquitoes were collected nationwide from a varietyof urban, periurban, and rural environments in theframework of the Portuguese National Program ofVector SurveillanceÐREVIVE (Alves et al. 2010). Trapsites included nonresidential areas such as rice (Oryzasativa L.) Þelds, marsh Þelds, water treatment plants,parks, and farms and residential areas in urban habitats(�411.5 inhabitants per km2 in 2010; http://www.ine.pt). In total, 43 sites in 34 counties belonging to 13districts in mainland Portugal were surveyed duringthe 2007Ð2010 mosquito seasons that in Portugal gen-erally last from April to October. In 2010, blood-fedmosquitoes were collected in residential areas of Ca-mara de Lobos and Funchal in the Autonomous Re-gion of Madeira Island from April to November. Baited(CO2) CDC light traps (John W. Hock Co., Gaines-ville, FL) were used according to Osorio et al. (2010).In addition, battery powered aspirators were used tosample Aedes aegypti L. mosquitos, particularly in theFunchal and Camara de Lobos residential areas. Mos-quitoes with fresh or visible blood remnants wereidentiÞed to species and individually transferred into1.5-ml microtubes and stored frozen at �80�C untilused for bloodmeal analysis (Ribeiro and Ramos1999).DNA Isolation From Blood-Fed Mosquitoes. Mos-

quito abdomens were removed with the aid of a ste-reomicroscope and reserved for bloodmeal analysis.Each mosquito was dissected individually on a newmicroscope slide by using ßame-sterilized forceps toavoid cross contamination. DNA was isolated from theabdominal contents of blood-fed mosquitoes individ-ually using DNeasy Blood & Tissue kit (QIAGEN

GmbH, Hilden, Germany), according to the manu-facturerÕs recommendations.Bloodmeal Analysis.To identify the vertebrate host

on which the mosquitoes had fed, polymerase chainreaction (PCR)Ðbased bloodmeal analyses were per-formed using a primer sequences for the cytochromeb published previously (Sorenson et al. 1999, Ciceroand Johnson 2001, Ngo and Kramer 2003, Molaei et al.2006). All DNA templates were initially screened withavian-speciÞc primers pairs a (5�-GAC TGT GACAAA ATC CCN TTC CA-3� and 5�-GGT CTT CATCTY HGG YTT ACA AGA C-3�) and mammalian-speciÞc primer pairs c (5�-CCA TCC AAC ATC TCAGCA TGA TGA AA-3� and 5�-GCC CCT CAG AATGAT ATT TGT CCT CA-3�). If the above-mentionedset of primers failed to yield any PCR product, threeadditionally universal primer sets were used: mamma-lian-a (5�-CGA AGC TTG ATA TGA AAA ACC ATCGTT G-3� and 5�-TGT AGT TRT CWG GT CHC CTA-3�), mammalian-b (5�-GCG TAC GCA ATC TTA CGATCA A-3� and 5�-CTG GCC TCC AAT TCA TGTGAG-3�), and avian-b (5�-CCC TCA GAA TGA TATTTGTCCTCA-3�andCCTCAGAAKGATATYTGNCCT CAK GG-3�). In some cases, these last primerswere used to solve ambiguous sequences. PCR andcycling conditions were as described by Molaei et al.(2006). High Fidelity PCR Master (Roche, Mann-heim, Germany) was used for all PCRs according tothe manufacturerÕs recommendations. Ampliconswere visualized by electrophoresis in a 1.5% agarosegel with GelRed (0.5Ð1 �g/ml) and puriÞed using JetQuick-PCR puriÞcation kit (Genomed, Lohne, Ger-many) as described by the manufacturer. The puriÞedDNA fragments were directly sequenced in an ABIautomated DNA capillary sequencer (Applied Biosys-tems, Foster City, CA) by using ABI Big Dye Termi-nator cycle sequencing ready reaction kit (AppliedBiosystems). Sequences were annotated by using Bi-oEdit software (Ibis Biosciences, Carlsbad, CA) andidentiÞed by comparison to the GenBank DNA se-quence database (National Center for BiotechnologyInformation, http://blast.ncbi.nlm.nih.gov/Blast.cgi).Positive identiÞcation and host species assignmentwere by sequence similarity. Sequences that did notmeet the criteria were reported as unknown. A blood-meal was classiÞed as mixed if two different specieswere identiÞed in two separate PCRs from the sametemplate or when chromatograms from each PCRdemonstrated double-nucleotide peaks. In addition,positive controls of known-origin blood were pro-cessed and correctly identiÞed with the above-men-tioned procedure. Species selected as controls in-cluded human (Homo sapiens), cow (Bos taurus), pig(Sus scrofa), and lizard (Lacerta dugesii).

Results

Bloodmeal sources were successfully identiÞed byDNA sequencing from 131 (64.5%) of 203 Þeld-col-lected mosquitoes with visible bloodmeals, represent-ing nine species of Þve genera. Of the remainingblood-fed mosquitoes, either PCR ampliÞcation was

718 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 49, no. 3

negative or the sequencing results were inconclusive.In total, 32 species of vertebrates were identiÞed,including 23 birds and nine mammals (Table 1).Cx. pipiens bloodmeals, which made up 78 of the

total bloodmeal sources sampled, were identiÞed mostcommonly as avian (n� 55/78; 70.5%), and less com-monly, but not infrequently, as mammals (n� 21/78;26.9%) in which humans were prevalent (n � 18/21;

85.7%), and sheep (Ovis aries) was the second mam-malian host detected (Table 2). Chicken (Gallus gal-lus) was the most common bird species identiÞed forCx. pipiens (n � 30/55; 54.6%), followed by commonblackbird (Turdus merula) (n� 5/55, 9.1%), and sev-eral other bird species frequent in Portugal. Mixedbloodmeals were detected in two specimens of Cx.pipiens, one including the blood of two mammal hosts,

Table 1. Bloodmeals by host class for mosquitoes’ species collected in Portugal, during 2007, 2008, 2009, and 2010 mosquito season

Species Mammalian host N Avian host N Mixed bloodmeal N

Ae. aegypti H. sapiens 6An. maculipennis B. taurus 5 Anser cygnoides 1

Capra hircus 1 G. gallus 1H. sapiens 2

Cs. annulata B. taurus 1H. sapiens 1O. aries 1

Cs. longiareolata H. sapiens 1Cx. perexiguus H. sapiens 1

O. aries 1Cx. pipiens H. sapiens 18 Annas platyrhyncos 1 H. sapiens/B. taurus 1

O. aries 3 Bradypterus tacsanowskius 1 H. sapiens/T. merula 1Carina moshata 1Cyanopica cooki 1Delichon urbica 1Falco tinnunculus 1Galerida cristata 1G. gallus 30Hyppolais polyglotta 1Laurus fuscus 1Meleagris gallopavo 1Miliaria calandra 1Nycticorax nycticorax 1Nymphicus hollandicus 1Parus caeruleus 1Passer domesticus 1Serinus canaria 1Sylvia borin 1Sylvia communis 2Sylvia melanocephala 1T. merula 5

Cx. theileri B. taurus 1 Morus bassanus 1 H. sapiens/B. taurus 1Canis familiaris 1Felis catus 1H. sapiens 3O. aries 1S. scrofa 2

Oc. caspius Canis familiaris 3Capra hircus 4Equus caballus 1H. sapiens 8Rattus rattus 1S. scrofa 1

Oc. detritus B. taurus 1O. aries 1

Table 2. Hosts of Cx. pipiens collected from residential and nonresidential areas of Portugal, during 2007, 2008, 2009, and 2010mosquito season

Habitat

Host

Avian

Mammal Mix

TotalHuman Sheep

Avian/Human

Bovid/Human

N % N % N % N % N % N %

Residential 10 66.7 5 33.3 0 0 0 0 0 0 15 19.2Nonresidential 45 71.4 13 20.6 3 4.8 1 1.6 1 1.6 63 80.8

Total 55 70.5 18 23.1 3 3.9 1 1.3 1 1.3 78 100

May 2012 OSORIO ET AL.: BLOODMEAL ANALYSIS OF MOSQUITOES IN PORTUGAL 719

human and bovid, and the other including human andavian blood from common blackbird (Tables 1 and 2).In relation to residential versus nonresidential habi-tats, no signiÞcant difference was detected inCx. pipi-ens blood-feeding behavior (P � 0.3; �2 test).

All the other species examined (Ae. aegypti, An.maculipennis, Cs. annulata, Cs. longiareolata, Cx. per-exiguus, Cx. theileri, Oc. caspius, and Oc. detritus) fedprimarily on mammals. Humans represented 100%(n � 6/6) of all the bloodmeals from Ae. aegypti andwas also the most common blood source identiÞed inOc. caspius(n� 8/18; 44.4%), which fed exclusively onmammals, as did Cs. annulata, Cs. longiareolata, Cx.perexiguus, and Oc. detritus. For An. maculipennis, 10avian and mammalian blood sources were identiÞed,including two bloodmeals from human. In one of 11tested specimens of Cx. theileri, a mixed bloodmealconsisting of human and bovid blood was detected(Table 1).

Discussion

In this study, six species (Ae. aegypti, Cs. annulata,Cs. longiareolata, Cx. perexiguus, Oc. caspius, and Oc.detritus) demonstrated an exclusive mammophilic-feeding behavior. The remaining analyzed species(An.maculipennis,Cx. pipiens, andCx. theileri) also fedon birds. Considering WNV epidemiology, these latestare the important species, with an ability to circulateand amplify WNV in nature and transmit the virus tohumans. WNV was isolated for the Þrst time in Por-tugal from An. maculipennis (Filipe and Pinto 1972).The avian and human bloodmeals detected in thisspecies support that it may play a role in WNV epi-demiology.Cx. pipiens is considered primarily ornithophilic

(Apperson et al. 2004, Turell et al. 2005), although itis clear from the data presented here that the blood-meals analyzed were not exclusively from birds. Theresults show that this species fed predominantly onavian hosts (70.5%), including some key bird speciesthat can maintain WNV transmission, in particular thecommon blackbird, house sparrows, and common kes-trel (Zeller and Schuffenecker 2004, Hayes et al.2005). This behavior supports the speculated signiÞ-cance of the role of Cx. pipiens in enzootic transmis-sion of WNV noted in previous studies (Molaei et al.2006; Hamer et al. 2008, 2009). However, 23.1% of allbloodmeals were human-derived, which points to Cx.pipiens not only as a potential enzootic vector but alsoas a potential bridge vector of WNV to humans. Themixed humanÐcommon blackbird bloodmeal con-Þrms the presence of individuals fromCx. pipiens pop-ulation readily feeding on both birds and humans, anecessary condition for epidemic transmission (Kil-patrick et al. 2005) and reinforces the role of Cx.pipiens in WNV epidemiology.

Regarding the Cx. pipiens identiÞed with humanbloodmeals, 27.8% (5/ 18) were collected from resi-dential areas as backyards of homes in urban habitatsand 72.2% (13/18) in mosquitoes collected in farms,natural parks, culture Þeld crops, and other rural and

periurban areas where humans are not, in any way, themost abundant hosts.Cx. pipienswas shown previouslyto be inßuenced by host availability (Hamer et al.2009). However, in this study no signiÞcant differ-ences were found between the proportions of avianÐhuman blood-fed Cx. pipiens in both residential andnonresidential habitats, although there was a smalldifference in variety of avian hosts used. Therefore,our Þnding of a high frequency of human feeding byCx. pipiensmosquitoes could not be explained by col-lecting blood-fed mosquitoes in proximity to humanhabitation. Some studies indicate that the innate hostpreference of many mosquito species is modulated bythe spatial and temporal abundances of potential hostsand by regional differences in host reproductive cy-cles and other behaviors (Richards et al. 2006, Savageet al. 2007); however, despite the limited number ofsamples in this study, it seems that there is no corre-lation between choice and availability of hosts. Forexample, the Cx. pipiens specimen that fed on a housesparrow was collected from a dog kennel. In this way,the results from this study support a clear innate hostpreference by Cx. pipiens that seemingly can be mod-ulated by spatial and temporal factors that were notevaluated. This preference can be supported, consid-ering that the two forms of the species Cx. pipiens,molestus, and pipiens, that occupy different habitats(underground versus aboveground) in northern Eu-rope can occur aboveground as sympatric populationsin southern Europe (Byrne and Nichols 1999, Huanget al. 2008, Kothera et al. 2010). It also has been sug-gested that the differential introgression of molestusgenes into the pipiens form may induce a more op-portunistic biting behavior in the latter (Gomes et al.2009). Hybrids betweenmolestus and pipiens are con-sidered epidemiologically signiÞcant because hybridscan readily feed on both avian and mammalian hosts,including humans. This opportunistic biting behaviormaypotentiate the roleofCx.pipiensas abridgevectorfor the transmission of arboviruses such as WNV fromtheir ampliÞcation hosts to humans (Fonseca et al.2004). Further investigation focusing on genetic datato characterized Cx. pipiens population in relation tofeeding habit is warranted.

Acknowledgments

We are grateful to Alice M. Nunes for technical assistancein data analyses. We also thank Portuguese National Programof Vector Surveillance (REVIVE) workgroup for the mos-quito collection. This work was supported by Centre forVector and Infectious Diseases Research/National Instituteof Health, Portugal.

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Received 19 August 2011; accepted 16 March 2012.

May 2012 OSORIO ET AL.: BLOODMEAL ANALYSIS OF MOSQUITOES IN PORTUGAL 721